Extracellular Vesicles Derived From Hypoxia-Conditioned Adipose-Derived Mesenchymal Stem Cells Enhance Lymphangiogenesis

Abstract

Extracellular vesicles from adipose-derived mesenchymal stem cells (ADSCs) play an important role in lymphangiogenesis; however, the underlying mechanisms are not fully understood. In this study, we aimed to investigate the function of extracellular vesicles secreted by hypoxia-conditioned ADSCs in lymphangiogenesis and explore the potential molecular mechanisms. Extracellular vesicles were extracted from ADSCs cultured under hypoxia or normoxia conditions. The uptake of extracellular vesicles by lymphatic endothelial cells (LECs) was detected by immunofluorescence staining. The effects of extracellular vesicles on the viability, migration, and tube formation of LECs were determined by CCK-8 assay, migration assay, and tube formation assay, respectively. Molecules and pathway involved in lymphangiogenesis mediated by ADSC-derived extracellular vesicles were analyzed by luciferase reporter assay, qRT-polymerase chain reaction (PCR), and Western blot. Hypoxia ADSC-derived extracellular vesicles (H-ADSC/evs) significantly enhanced the proliferation, migration, and tube formation of LECs. Hypoxia decreased the expression of miR-129 in ADSC-derived extracellular vesicles. Overexpression of miR-129 counteracted the promoting effect of H-ADSC/evs on lymphangiogenesis. In addition, decreased exosomal miR-129 expression resulted in upregulation of HMGB1 in LECs, which led to AKT activation and lymphangiogenesis enhancement. Our data reveal that extracellular vesicles derived from hypoxia-conditioned ADSCs induce lymphangiogenesis, and this effect is mediated by miR-129/HMGB1/AKT signaling. Our findings imply that hypoxia ADSC-isolated extracellular vesicles may represent as a valuable target for the treatment of diseases associated with lymphatic remodeling.

Introduction

Lymphangiogenesis is a process that forms new lymphatic vessels from pre-existing lymphatic vasculature and plays important roles in many diseases, such as cancer, inflammatory bowel disease, and endometriosis^1–3. Mesenchymal stem cell (MSC)-based therapy has been considered as a promising strategy for lymphatic regeneration ⁴ , but the underlying mechanism is not fully clarified.

Accumulating evidence suggests that MSCs exert their function via secreting vesicles and molecules^5,6. Extracellular vesicles are cellular vesicles (40–100 nm in diameter) that are secreted by different types of cells. Extracellular vesicles play an critical function in many processes, such as cell proliferation, survival, migration, apoptosis, immune response, by delivering proteins, mRNAs, or miRNAs from host cells to recipient cells^7,8. Adipose-derived mesenchymal stem cells (ADSCs) are a population of pluripotent cells derived from adipose tissue, whose characteristics are similar to bone marrow-derived mesenchymal stem cells (BMSCs) ⁹ . Multiple studies have reported that ADSCs have potential application for promoting lymphatic generation^10–12. It has been reported that ADSCs promote VEGF-C-dependent lymphangiogenesis via secreting extracellular vesicles ⁶ , indicating that the paracrine effect is critical for ADSC-mediated lymphangiogenesis promotion.

The oxygen concentration is an important factor for stem cell proliferation and differentiation. In the body, the oxygen content is quite low in adipose tissue and thus ADSCs usually exist in low oxygen conditions ¹³ . Consistently, treatment with hypoxia can promote the proliferation and differentiation of ADSCs and alter their molecule production ¹⁴ . However, the function of extracellular vesicles from hypoxia-conditioned ADSCs in lymphangiogenesis remain unclear. In the current study, we investigated the effects of hypoxia ADSC-derived extracellular vesicles on lymphatic endothelial cell proliferation, migration and tube formation, and explored the molecules and pathway involved in this process.

Materials and Methods

Cell Culture

Human ADSCs and dermal lymphatic endothelial cells (LECs) were obtained from Guangzhou Cyagen Biology (Huangpu District, Guangzhou, China). ADSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) under hypoxia (1% O₂) or normoxia conditions (21% O₂). LECs were maintained in endothelial growth medium-2-MV (EGM-2-MV, Lonza) that contains endothelial basal medium-2 (EBM-2, Lonza) and SingleQuots kit (Lonza).

Extracellular Vesicles Isolation

ADSCs were cultured under hypoxia or normoxia conditions for 24 h, and the medium was harvested for extracellular vesicles isolation. The medium was centrifuged at 3,000 g for 15 min and then 20,000 g for 45 min at 4°C. The supernatant was filtered and further centrifuged at 110,000 g for 70 min at 4°C. The sample were washed once with phosphate buffered saline (PBS) and then recentrifuged at 110,000 g for another 70 min at 4°C.

Transmission Electron Microscopy

Extracellular vesicles isolated from ADSCs were resuspended in PBS. A drop of extracellular vesicles was placed on the top of a copper grid. After washing once with PBS, extracellular vesicles were fixed with 3% glutaraldehyde for 15 min. The sample was washed again with PBS and incubated with 2% uranyl acetate for 5 min. Then, exosomes were visualized under a transmission electron microscope. Dynamic light scattering was used to determine the exosome size.

Uptake of Extracellular Vesicles

ADSC-derived extracellular vesicles were labeled with KH67 dye (Sigma, USA). LECs were seeded in 6-well plates and 2 μg KH67-stained extracellular vesicles were added. After incubation for 24 h, LECs were fixed with 4% paraformaldehyde. After stained with DAPI, LECs were analyzed under a confocal microscope (Carl Zeiss, Germany), and the location of extracellular vesicles was visualized.

CCK-8 Assay

LECs (2,000 cells/well) in 100 μl medium were seeded in 96-well plate. For detecting the viability of LECs, 10 μl Cell Counting Kit-8 (CCK-8) reagent (Seyotin, China) was added to each well. After incubation for 1~4 h, the absorbance at 450 nm was determined by a plate reader.

Transwell Migration Assay

LECs (1× 10⁵) in 100 μl serum-free medium were added into Transwell inserts (Corning, USA), and 600 μl completed medium with 10 μg/ml extracellular vesicles was added to the lower compartment of the chamber. After incubation for 24 h, LECs were fixed with methanol for 10 min and stained with 0.0.5% crystal violet for 15 min. The non-migrating cells were scraped with a cotton swab, and the migrated cells were visualized under an inverted microscope (Carl Zeiss, Germany).

Tube Formation Analysis

Twenty-four-well plates were precoated with Matrigel (BD Sciences, USA) for 30 min at 37°C prior to cell seeding. LECs (2×104 cells/well) were seeded in 24-well plates and 10 μg/ml ADSC-derived extracellular vesicles were added. After incubation for 24 h, the tube number at three random field was counted using a light microscope.

Quantitative RT-PCR

LECs were treated with ADSC-derived extracellular vesicles (10 μg/ml) for 24 h. LECs were collected and used for RNA isolation. Total RNA was extracted using TRIzol reagent (Life Science, Waltham, MA, USA) and was reverse transcribed to cDNA using the PrimeScript RT-PCR kit (Seyotin, China). qPCR was performed with the qPCR Mix (Seyotin, china) on the Applied Biosystems 7300 system (Applied Biosystems, USA). The experiments were conducted in triplicate. Gene (or miRNA) expression was normalized to GAPDH (or U6) using the 2^-ΔΔCT formula.

DNA and RNA Transfection

miR-129 mimics and control mimics (NC mimics) were purchased from RiboBio (Guangzhou, China). HMGB1 expression plasmid and control plasmid were obtained from Hanbio (Shanghai, China). miRNA mimics and HMGB1 expression plasmid were transfected into LECs using Lipofectamine 2000 (ThermoFisher, USA) according to the manufacturer’s instructions.

Luciferase Reporter Assay

The wild type and mutated HMGB1 3’UTR were cloned into pGL3 plasmid to form recombinant plasmids pGL3-HMGB1-3’UTR-wt and pGL3-HMGB1-3’UTR-mut. LECs (2 × 105 cells/well) were seeded in 24-well plates and were cotransfected with pGL3-HMGB1-3’UTR-wt (or pGL3-HMGB1-3’UTR-mut), miR-129 mimics (or NC mimics), and pRL-TK (Promega, USA). After transfection for 48 h, the luciferase activity was determined by using a Dual-Luciferase Reporter Assay System (Promega).

Western Blot

LECs or ADSC-derived extracellular vesicles were lysed using RIPA lysis buffer. The sample was separated by SDS-PAGE gel and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, the PVDF membrane was incubated with primary antibody at 4°C overnight. After washing three times with TSB-Tween 20 (TBST), the membrane was incubated with the secondary antibody for 1.5 h at room temperature. After three washes with TBST, the protein bands were detected by using the Western Blot Detection Kit (Seyotin, China). The antibodies used were as follows: CD9 (ab59479; Abcam, UK), CD63 (ab59479; Abcam), TSG-101 (ab125011; Abcam, UK), HMGB1 (ab18256; Abcam, UK), LYVE-1 (ab14917; Abcam, UK), PROX1 (ab101851; Abcam, UK), GAPDH (#2118; Cell Signaling Technology, USA), AKT (#9272; Cell Signaling Technology, USA), and p-AKT (#4060; Cell Signaling Technology, USA).

Statistical Analysis

The data were represented as the mean ± standard deviation (SD) from three independent experiments. Statistical comparisons were performed with Student t-test (two groups) or one-way analysis of variance (ANOVA; more than two groups). A P value lower than 0.05 was considered as statistically significant.

Results

Characterization of Extracellular Vesicles Derived From ADSCs

To identify the potential activity of MSC-derived extracellular vesicles on lymphangiogenesis, exosomes were isolated from ADSCs cultured under hypoxia (H-ADSC/evs) and normoxia conditions (N-ADSC/evs). Transmission electron microscopy and nanoparticle tracking analysis showed that extracellular vesicles were spherical and of 100~150 nm diameter and the morphology and size of H-ADSC/evs and N-ADSC/evs were similar (Fig. 1A, B). To further confirm the extracellular vesicles, the expression of exosomal markers (CD9, CD63, and TSG-101) were analyzed by Western blot. The expression of CD9, CD63, and TSG-101 was detected in both H-ADSC/evs and N-ADSC/evs (Fig. 2C). Next, the internalization of exosomes by LECs were investigated by immunofluorescence staining. The result showed that PKH67-labeled extracellular vesicles could be taken up by LECs (Fig. 1D).

Figure 1.

Identification of hypoxia ADSC-derived extracellular vesicles. (A) Transmission electron microscopy analysis of the morphology of H-ADSC/evs and N-ADSC/evs. Scale bars: 100 nm. (B) NTA analysis of H-ADSC/exos and N-ADSC/exos. (C) Western blot analysis of CD9, CD63, and TSG-101 expression in of H-ADSC/evs and N-ADSC/evs. (D) Immunofluorescence staining determining the uptake of hypoxia ADSC-derived extracellular vesicles by LECs. Data represent the mean ± SD of three separate experiments; comparison was performed with Student’s t-test. Scale bar: 100 μm. ADSCs: adipose-derived mesenchymal stem cells; H-ADSC/evs: hypoxia ADSC-derived extracellular vesicles; LECs: lymphatic endothelial cells; N-ADSC/evs: normoxia ADSC-derived extracellular vesicles; SD: standard deviation.

Figure 2.

Effects of hypoxia ADSC-derived extracellular vesicles on LEC proliferation, migration, and tube formation. (A) CCK-8 assay in LECs after treatment with or without H-ADSC/evs (or N-ADSC/evs). (B) Migration assay in LECs after treatment with or without H-ADSC/evs (or N-ADSC/evs). (C) Tube formation assay in LECs after treatment with or without H-ADSC/evs (or N-ADSC/evs). (D, E) qRT-PCR and Western blot analysis of LYVE-1 and PROX1 expression in LECs after treatment with or without H-ADSC/evs (or N-ADSC/evs). *P < 0.05, **P < 0.01. Data represent the mean ± SD of three separate experiments; comparison was performed with Student’s t-test. Scale bar: 100 μm. ADSCs: adipose-derived mesenchymal stem cells; CCK-8: Cell Counting Kit-8; H-ADSC/evs: Hypoxia ADSC-derived extracellular vesicles; LECs: lymphatic endothelial cells; N-ADSC/evs: normoxia ADSC-derived extracellular vesicles; mRNA: messenger RNA; SD: standard deviation.

H-ADSC/evs Increases LEC Proliferation, Migration, and Tube Formation

To investigate whether H-ADSC/evs could influence lymphangiogenesis, we detected the effects of H-ADSC/evs on the proliferation, migration, and tube formation in LECs. After treatment with H-ADSC/evs and N-ADSC/evs, the viability of LECs were analyzed by CCK-8. The result showed that H-ADSC/evs significantly increased the viability of LECs compared with N-ADSC/evs (Fig. 2A). Migration assay also showed that treatment with H-ADSC/evs highly elevated the migration activity of LECs compared with N-ADSC/evs (Fig. 2B). In addition, tube number was significantly increased in H-ADSC/evs-treated group compared with the N-ADSC/evs-treated group (Fig. 2C). Moreover, H-ADSC/evs highly increased the mRNA and protein expression of lymphangiogenesis modulators, LYVE-1 and PROX1, compared with N-ADSC/evs (Fig. 2D, E). Together, these data indicate that H-ADSC/evs has a positive effect on LEC proliferation, migration, and tube formation.

Hypoxia-Conditioned ADSC/evs Promote Lymphangiogenesis by Reducing miR-129

Given the important role of miRNAs in lymphangiogenesis, we tested the expression of several miRNAs in extracellular vesicles from ADSCs under hypoxia or normoxia conditions. The result showed that miR-129 was significantly decreased in H-ADSC/evs compared with N-ADSC/evs, while other miRNAs were not altered obviously (Fig. 3A). To access the contribution of miR-129 to H-ADSC/evs-mediated lymphangiogenesis, we transfected miR-129 mimic to LECs under hypoxia or normoxia conditions before extracellular vesicles extraction. Cell viability analysis and Transwell migration assay showed that treatment with miR-129 decreased the viability and migration of LECs that were increased by H-ADSC/evs (Fig. 3B, C). Additionally, tube formation assay revealed that H-ADSC/evs increased tube formation of LECs, and transfection of miR-129 mimics reversed this effect (Fig. 3D). Collectively, these results suggest that H-ADSC/evs enhances lymphangiogenesis via reduction of miR-129.

Figure 3.

H-ADSC/evs increase lymphangiogenesis by downregulation of miR-129. (A) qRT-PCR analysis of miRNA expression in LECs treated with H-ADSC/evs or N-ADSC/evs. (B) CCK-8 assay in LECs after treatment with H-ADSC/evs (or N-ADSC/evs) together with miR-129 mimics (or NC mimics). (C) Transwell migration assay in LECs after treatment with H-ADSC/evs (or N-ADSC/evs) together with miR-129 mimics (or NC mimics). (D) Tube formation assay in LECs after treatment with H-ADSC/evs (or N-ADSC/evs) together with miR-129 mimics (or NC mimics). Data represent the mean ± SD of three separate experiments; comparison was performed with Student’s t-test. Scale bar: 100 μm. ADSCs: adipose-derived mesenchymal stem cells; CCK-8: Cell Counting Kit-8; H-ADSC/evs: Hypoxia ADSC-derived extracellular vesicles; LECs: lymphatic endothelial cells; N-ADSC/evs: normoxia ADSC-derived extracellular vesicles; mRNA: messenger RNA; SD: standard deviation. *P < 0.05, **P < 0.01.

miR-129 Suppresses H-ADSC/evs-Mediated Lymphangiogenesis via HMGB1

miRNAs exert their functions usually via modulation of downstream target genes. HMGB1 has been reported to play an important role in lymphangiogenesis and its association with miR-129 has been indicated previously ¹⁵ . Our data showed that treatment with H-ADSC/evs increased the expression of HMGB1, and this effect was blocked by overexpression of miR-129 (Fig. 4A). Luciferase reporter assay showed that transfection with miR-129 mimics could reduce the luciferase activity of HMGB1 3’UTR, but had no significant effect on HMGB1 3’UTR mutant in LECs (Fig. 4B, C), indicating that HMGB1 is a direct target of miR-129 in LECs. Next, we investigated whether miR-129 inhibited lymphangiogenesis via regulating HMGB1. CCK-8 assay showed that miR-129 suppressed H-ADSC/evs-promoted LEC viability and this effect was reversed by overexpression of HMGB1 (Fig. 4D). Similarly, while miR-129 reduced the migration and tube formation of LECs that were promoted by H-ADSC/evs, ectopic expression of HMGB1 counteracted these effects of miR-129 (Fig. 4E, F). Collectively, these data suggest that H-ADSC/evs promotes lymphangiogenesis via modulating miR-129/HMGB1 cascade.

Figure 4.

Exosomal miR-129 regulates HMGB1 expression. (A) Western blot analysis of HMGB1 expression in LECs after treatment with H-ADSC/evs with miR-129 mimics or NC mimics. (B) The binding site of miR-129 in HMGB1 3’UTR. (C) Luciferase reporter assay in LECs transfected with miR-129 and wild type (wt) or mutated (mut) HMGB1 3’UTR. (D) CCK-8 assay demonstrated that transfection of miR-129 abolished the effect of H-ADSC/evs on LEC viability, and this effect was reversed by overexpression of HMGB1. (E) Transwell migration assay revealed that transfection of miR-129 mimics counteracted the effect of H-ADSC/evs on LEC migration, and this effect was reversed by overexpression of HMGB1. (F) Tube formation assay showed that overexpression of miR-129 restrained the effect of H-ADSC/evs on LEC tube formation, and this effect was reversed by transfection of HMGB1. Data represent the mean ± SD of three separate experiments; comparison was performed with Student’s t-test. Scale bar: 100 μm. ADSCs: adipose-derived mesenchymal stem cells; CCK-8: Cell Counting Kit-8; H-ADSC/evs: Hypoxia ADSC-derived extracellular vesicles; LECs: lymphatic endothelial cells; N-ADSC/evs: normoxia ADSC-derived extracellular vesicles; SD: standard deviation. *P < 0.05, **P < 0.01.

HMGB1 Induces Lymphangiogenesis via Activation of AKT

To understand the possible pathway by which HMGB1 induced lymphangiogenesis, we tested the expression and phosphorylation of AKT, which is implicated in lymphangiogenesis. Western blot analysis showed that treatment with H-ADSC/evs markedly increased the phosphorylation of AKT, but had no obvious effect on its protein level (Fig. 5A), indicating that H-ADSC/evs could activate AKT signaling. To confirm the involvement of the AKT pathway in H-ADSC/evs-mediated lymphangiogenesis, LECs were treated with AKT inhibitor perifosine. Transwell migration assay showed that H-ADSC/evs increased LEC migration, and treatment with perifosine restrained this promotion (Fig. 5B). Also, administration of perifosine decreased the tube number which was increased by H-ADSC/evs. Taken together, these data indicate that H-ADSC/evs enhances lymphangiogenesis via HMGB1-mediated activation of AKT signaling.

Figure 5.

Hypoxia-conditioned ADSC-derived extracellular vesicles activates AKT via HMGB1. (A) Western blot analysis of AKT expression and phosphorylation in LECs after treatment with H-ADSC/evs and transfection with HMGB1 siRNA (si-HMGB1) or control siRNA (si-NC). (B) Treatment with perifosine (30 μM) decreased the tube formation of LECs enhanced by H-ADSC/evs. (D) Treatment with perifosine (30 μM) reduced H-ADSC/evs-induced LYVE-1 and PROX1 expression. Data represent the mean ± SD of three separate experiments; comparison was performed with Student’s t-test. Scale bar: 100 μm. ADSCs: adipose-derived mesenchymal stem cells; H-ADSC/evs: Hypoxia ADSC-derived extracellular vesicles; LECs: lymphatic endothelial cells; N-ADSC/evs: normoxia ADSC-derived extracellular vesicles; SD: standard deviation. *P < 0.05, **P < 0.01.

Discussion

Lymphangiogenesis is associated with many critical biological progresses. Increasing reports suggest that MSC-derived extracellular vesicles are potential targets for lymphangiogenesis. However, the underlying mechanisms remain largely unclear. In the present study, we confirmed the promoting effect of extracellular vesicles derived from ADSCs under hypoxia conditions on lymphangiogenesis. Importantly, our results revealed that hypoxia ADSC-derived extracellular vesicles induced lymphangiogenesis via activation of AKT signaling through miR-129/HMGB1 cascade.

A number of miRNAs have been reported to play an important role in lymphangiogenesis ¹⁶ . For example, it has been reported that miR-155 promotes lymphatic generation by activating STAT3/VEGFC signaling ¹⁷ . MiR-199a/b-5p suppresses lymphangiogenesis by modulating DDR1 ¹⁸ . miR-129 has been reported to play crucial roles in multiple processes, including cell proliferation, migration, apoptosis, and angiogenesis^19–21. It has been also reported that miR-129 inhibits lymphangiogenesis in nasopharyngeal cancer via targeting ZIC2 ²² . In the present study, we found that the expression of miR-129 was downregulated in H-ADSC/exos-treated LECs compared with N-ADSCs/exos-treated LECs. Moreover, transfection of miR-129 could block the promoting effect of H-ADSC/evs on LEC proliferation, migration, and tube formation. Therefore, our data indicate that the decreased miR-129 expression contributes to H-ADSC/evs-induced lymphatic generation.

HMGB1 is a heparin-binding protein that is involved in many biological processes, such as cell growth, survival, apoptosis, migration, immune response, and so on^23–26. Mutiple studies have demonstrated that HMGB1 also has an important role in lymphagiogenesis. For instance, it has been reported that curcumin suppresses the lymphangiogenesis of gastric cancer cells via suppression of HMGB1/VEGF-D signaling ²⁷ . Han et al. ²⁸ reported that HMGB1 enhances inflammation-mediated lymphangiogenesis through TLR4-dependent signaling pathway. Li et al. ²⁹ also showed that HMGB1 activates NF-kB to upregulate VEGF-C and thus promotes lymphangiogenesis and lymphatic node metastasis in colon cancer. Consistently, in our study, we found that HMGB1 was a direct target of miR-129 in LECs. Moreover, downregulation of exosomal miR-129 resulted in increased HMGB1 expression in LECs, which consequently led to elevated LEC migration and tube formation.

Accumulating evidence suggests that the AKT-mediated signaling participates in lymphangiogeneis. It has been reported that Qingjie Fuzheng Granule inhibits lymphangiogenesis via the VEGF-C/VEGFR-3-dependent AKT pathway in colorectal cancer ³⁰ . Treatment with human cathelicidin antimicrobial peptide LL-37 enhances lymphangiogenesis in lymphatic endothelial cells through the AKT and ERK signaling pathways ³¹ . Previous studies have been demonstrated that HMGB1 is an upstream regulator of AKT signaling^32,33. Here, we found that treatment with H-ADSC/evs increased AKT phosphorylation, and this effect was abolished by HMGB1 silencing. Moreover, treatment with AKT inhibitor perifosine blocked the promoting effects of H-ADSC/evs on LEC migration, as well as the expression levels of LYVE-1 and PROX1. Therefore, HMGB1/AKT signaling is linked to the function of H-ADSC/evs in lymphangiogenesis.

Conclusion

Our results showed that extracellular vesicles secreted by ADSCs cultured under hypoxia conditions have elevating effects on LEC proliferation, migration, and tube formation. Hypoxia reduced the expression of miR-129 in ADSC-derived extracellular vesicles, which led to activation of HMGB1/AKT signaling and thus enhance lymphangiogenesis. Therefore, hypoxia ADSC-secreted extracellular vesicles may serve as a novel strategy for the therapy of lymphangiogenesis-associated diseases.